The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PDGFR alpha / beta. The final product is generated by affinity chromatography using a PDGFR alpha derived peptide phosphorylated at Tyrosines 572 and 574.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

Use a concentration of 0.35 - 1 µg/ml. Detects a band of approximately 170 - 185 kDa.

Target

Relevance

Platelet derived growth factor receptor (PDGFR) is a transmembrane glycoprotein of 170-185 kDa which undergoes homo- or heterodimerization into complexes of alpha and beta subunits upon ligand binding, depending on the isoform of PDGF (PDGF-AA, -BB or -AB) that binds. The phosphorylation of tyrosine residues in the now activated receptor can control multiple signaling events such as actin reorganization, transcription, cell growth, migration and differentiation. PDGFR alpha tyrosines 572 and 574 (579 and 581 in PDGFR beta) are autophosphorylated in the activated receptor, and bind and activate Src family kinases.

Images

Peptide Competition: Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.50 µg/mL ab5443 antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab5443 blocks the antibody signal, thereby demonstrating the specificity of the antibody. Peptide Competition: Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF